CN111024654A - Preparation method of optical fiber sensor and application of optical fiber sensor in bacterial detection - Google Patents
Preparation method of optical fiber sensor and application of optical fiber sensor in bacterial detection Download PDFInfo
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Abstract
The invention discloses an omega-shaped optical fiber detection probe, which is combined with biosensing to obtain a novel sensor for detecting pathogenic bacteria. The novel omega-shaped optical fiber is processed by adopting flame at high temperature, and the processing process is simple. The FOLSPR probe is assembled by modifying the surface of an optical fiber and connecting nanogold to prepare optical fiber surface plasmon resonance (FOLSPR), connecting aptamer DNA on the surface of the FOLSPR and adopting continuous spectrum, thereby realizing the real-time detection of a biological sample. The performance of the probe is researched, the advantages of the refractive sensitivity of the optical fiber probe are demonstrated, the mechanism causing the sensitivity to be improved is deeply researched, and finally the probe is successfully applied to the detection of pathogenic bacteria. The novel omega-shaped optical fiber sensor has the advantages of simplicity and convenience in operation, high sensitivity, good stability and capability of detecting biological combination in real time.
Description
Technical Field
The invention relates to a preparation method of an optical fiber sensor and application of the optical fiber sensor in bacterial detection.
Background
Salmonella, a commonly reported gastrointestinal infectious bacterium in humans and animals, is often associated with food-borne disease outbreaks. Salmonella typhimurium is food borne and has a high mortality rate. In addition, it has strong resistance in external environment and can be rapidly propagated at normal temperature. Therefore, the development of a sensitive and accurate detection and analysis method is of great significance to public health and food safety.
Fiber Localized Surface plasmon resonance (FOLSPR) has the advantages of no signal loss, long-distance transmission, low cost, no label, real-time detection, simple installation and the like. However, FOLSPR exhibits relatively low sensitivity compared to conventional fiber surface plasmon resonance (folpr), which hinders further development of this technology. Varying the geometry can change the Refractive (RT) sensitivity of the FOLSPR. However, the currently used D-type optical fiber, S-type optical fiber and tapered optical fiber all face the problem of difficult processing, and also have the problems of large requirements for sample amount, difficult manufacturing of sample cell, and the like, which is not very favorable for the application in the research of sensors. The U-shaped optical fiber is easy to process, relatively high in sensitivity and small in sample requirement, and is often applied to the research of sensors.
Here we describe a new FOLSPR sensor which is simple to manufacture, small in sample volume, simple in sample cell, and the most important feature is a refractive sensitivity significantly higher than that of the U-shaped fiber. Meanwhile, the reason for high refraction sensitivity of the novel FOLSPR sensor is explored, the real-time, label-free, high-sensitivity and specific detection of pathogenic bacteria is realized, and the method has important significance on public health and food safety.
Disclosure of Invention
The invention provides a preparation method of a novel optical fiber surface plasma resonance sensor, and the prepared novel optical fiber surface plasma resonance sensor is applied to the high-sensitivity, high-specificity, real-time and label-free detection of pathogenic bacteria. The kit realizes high sensitivity, high specificity, real-time and label-free detection on pathogenic bacteria.
The invention adopts the technical scheme that the invention achieves the aim that:
the invention provides an optical fiber, which has the following structure: the cladding-containing part 2 is characterized by comprising an exposed part 1 and a cladding-containing part 2, wherein the exposed part 1 comprises an arc section 3 larger than a semicircle and two straight sections 4, one ends of the two straight sections 4 are respectively connected with two ends of the arc section 3, the connecting part of the straight sections 4 and the arc section 3 is rounded, the distance between the two straight sections 4 is gradually increased from one end connected with the arc section 3 to the other end, and the other end of the straight section 4 is connected with the cladding-containing part 2; preferably, the core diameter of the optical fiber is 600 μm, and the length of the optical fiber is 10-50 cm.
Exposed parts: only the core of the fiber, not the cladding;
portion containing cladding: a core comprising an optical fiber and a cladding surrounding the surface thereof.
The invention also provides a preparation method of the optical fiber, which comprises the following steps: taking an optical fiber, polishing two ends of the optical fiber to a smooth and flat state, burning off a cladding of a part needing to be exposed under the condition of high temperature of flame, and processing and molding.
The invention also provides application of the optical fiber in preparing the optical fiber surface plasma resonance probe.
The invention also provides an optical fiber surface plasma resonance probe, wherein an adapter body for detection is connected to the surface of the exposed part of the optical fiber; preferably, the aptamer is a thiol-modified aptamer; more preferably, the thiol-modified aptamer is: SH-C6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG.
The invention also provides a preparation method of the optical fiber surface plasma resonance probe, which comprises the following steps:
1) the bare portion of the optical fiber was immersed in 30% H2O2And concentrated H2SO4Ultrasonic washing and drying are carried out in the mixed solution;
2) then immersing the bare part of the optical fiber into a 3-APTMS solution, cleaning and drying;
3) then immersing the bare part of the optical fiber into the nano gold solution, modifying for 10-40 minutes, preferably 15 minutes, and drying;
4) finally, connecting a mercapto-modified aptamer on the surface of the exposed part of the optical fiber to obtain an optical fiber surface plasma resonance probe;
preferably, the first and second electrodes are formed of a metal,
in step 1), 30% H2O2With concentrated H2SO4The volume ratio of the components is 3: 7, the soaking temperature is 90 ℃, and the soaking time is 30 minutes;
in the step 2), the preparation method of the 3-APTMS solution is to dissolve the 3-APTMS in a mixture of ethanol and acetic acid with the volume ratio of 5: 2, and the concentration of the 3-APTMS solution is 1 percent (v/v);
in the step 2), the mixture is subjected to ultrasonic cleaning for 15 minutes and water cleaning for 10 minutes in 3-APTMS solution at room temperature for 10-20 minutes, preferably 15 minutes;
in the step 3), the synthesis method of the nano gold is to mix 50mL of HAuCl in a flask4·3H2Heating the O aqueous solution to boiling, adding a trisodium citrate solution into the flask, continuing to boil the mixture for 15 minutes when the red color of the wine is stable, and cooling to room temperature under stirring; wherein, HAuCl4·3H2The concentration of O is 0.1mg/mL, and the concentration of trisodium citrate solution is 1% (w/v);
in the step 3), the soaking conditions in the nano gold solution are as follows: at room temperature, 10-40min, preferably 15min, and drying directly without cleaning.
The invention also provides application of the optical fiber surface plasma resonance probe in preparing an optical fiber surface plasma resonance probe sensor.
The invention also provides an optical fiber surface plasma resonance probe sensor which comprises the optical fiber surface plasma resonance probe, a tungsten lamp light source and an optical fiber spectrometer.
The invention also provides application of the optical fiber surface plasma resonance probe sensor in pathogenic bacteria detection.
Further, the pathogenic bacteria are salmonella typhimurium, staphylococcus aureus, shigella, escherichia coli and salmonella enteritidis; preferably, the salmonella typhimurium concentration is changed to 5 × 102To 1X 108CFU/mL, Staphylococcus aureus, Shigella, Escherichia coli, Salmonella enteritidis, as control for specific recognition, at a concentration of 106CFU/mL。
Further, the binding time of the pathogenic bacteria and the aptamer is 1h-3h, and preferably 1.5 h;
and/or the culture conditions of pathogenic bacteria are as follows: LB medium, 200rpm/min, 37 ℃ overnight, the temperature for pathogen and aptamer binding was 37 ℃.
The novel omega-shaped FOLSPR probe sensor provided by the invention has the process characteristics that the sensor comprises the following steps:
1. the preparation method of the omega-shaped FOLSPR probe sensor comprises the following steps:
1) preparing an optical fiber; a communication fiber having a core diameter of 600 μm is used, and the length thereof is 10 to 50cm, preferably 20 cm. Polishing the two ends of the optical fiber to a smooth and flat state, burning off the cladding under the condition of flame high temperature, and processing into an omega shape.
2) Functionalization of the optical fiber; the optical fiber is subjected to surface modification to achieve the purpose of detection. Optical fiber depacketized partial immersion H2O2And H2SO4And (4) mixing the solution. Ultrasonic washing, drying, immersing in 3-APTMS solution, washing and drying. Soaking in nanogold (AuNPs) solution to obtain FOLSPR.
30% H in step 2)2O2With concentrated H2SO4The volume ratio of (A) to (B) is 3: 7, and the reaction temperature is 90 ℃.
The 3-APTMS solution in the step 2) has the concentration of 1% (v/v) and is prepared by dissolving 3-APTMS in a mixture of ethanol and acetic acid in a volume ratio of 5: 2.
After the 3-APTMS solution is soaked in the step 2), the cleaning condition is that 95% ethanol is used for ultrasonic cleaning for 15 minutes, and the water is used for 10 minutes.
3) Step 2) the AuNPs synthesis method comprises the following steps: 50mL HAuCl in flask4·3H2The aqueous O solution was heated to boiling and then the flask was charged with a solution of trisodium citrate. When the wine red was stable, the mixture was boiled for a further 15 minutes and cooled to room temperature with stirring.
4) Step 3) the concentration of HAuCl4 & 3H2O is 0.1mg/mL, and the concentration of trisodium citrate solution is 1% (w/v).
5) And connecting the sulfydryl modified aptamer on the surface of the FOLSPR, and functionalizing the FOLSPR probe.
6) The concentration of the aptamer used in step 5) was 1. mu.M. Aptamer DNA was dissolved in buffer solution and FOLSPR was immersed therein for modification.
7) The buffer solution in the step 6) comprises the following components: 10mM Tris-HCl pH 7.4, 5mM KCl, 100mM NaCl, 1mM MgCl2
2. Use of an omega-shaped FOLSPR probe sensor for bacterial detection.
1) Bacteria were detected with a functionalized omega-shaped FOLSPR probe to analyze the sensitivity, selectivity and range of application of the FOLSPR sensor.
2) The aptamer and the bacterium in step 1) are specifically recognized.
3) The aptamer in the step 2) is specifically identified by the typhimurium, and has the sequence: SH-C6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG. The bacteria involved were: salmonella typhimurium, Staphylococcus aureus, Shigella, Escherichia coli, Salmonella enteritidis.
4) The concentration change of the salmonella typhimurium in the step 3) is 5 multiplied by 102To 1X 108CFU/mL. Other bacteria as specific recognitionControl, concentration 106CFU/mL。
5) The binding time of the bacteria and the aptamer in the step 4) is 1h-3h, and preferably 1.5 h.
6) The bacterial culture conditions in the step 5) are as follows: LB medium, 200rpm/min, 37 ℃ overnight. Binding conditions for FOLSPR: 37 ℃ is carried out.
7) The above detection results show that the concentration is 5X 102To 1X 108The absorbance in the CFU/mL range is linear with log change of the bacteria and is not combined with other bacteria. Exhibits a wide detection range and high specific binding.
The above FOLSPR sensor of the present invention can be implemented by the following detection means.
The implementation device of the FOLSPR sensor is characterized in that the collected spectrum is a continuous spectrum, and the reaction process can be monitored in real time. The FOLSPR probe is used as a center, the input end of the FOLPSR probe is connected to a tungsten lamp light source, the output end of the FOLPSR probe is connected to a fiber spectrometer, and signals are output to a computer. The reaction temperature is controlled by the metal bath and the up and down movement of the FOLSPR is controlled by the displacement table.
Compared with the existing FOLSPR technology, the omega-shaped FOLSPR sensor provided by the invention has the following outstanding beneficial technical effects and advantages:
1. because the omega-shaped optical fiber is simple to process, compared with optical fibers with other shapes, such as D-shaped optical fibers, S-shaped optical fibers and the like, the omega-shaped optical fiber is simpler to manufacture, has small sample amount and is simple in sample cell. The cladding can be removed by high-temperature burning of the alcohol burner, and the product is processed into the required shape. The sample cell is a common centrifuge tube, so the cost is low, and special processing is not needed. Due to the small volume of the fiber optic probe, minimal sample measurements can be made in a 200 μ L centrifuge tube.
2. The omega-shaped optical fiber of the present invention has a unique shape with more bending area than the most commonly used U-shaped optical fiber. The U-shaped optical fiber only has a bending area which is approximately semicircular, and the omega-shaped optical fiber has a bending area which is approximately full of circle, so that the U-shaped optical fiber has higher refraction sensitivity.
3. The bending region has a decisive influence on the enhancement of the refraction sensitivity of the FOLSPR, and the higher the refraction sensitivity, the higher the detection sensitivity of the FOLSPR probe is. Therefore, the bending region has a large influence on the performance of the FOLSPR. However, it is not necessarily said that the more bending regions are better, for example, the 8-shaped and spiral-shaped bending regions have more bending regions than the omega-shaped bending regions, but by monitoring the time curve of the signal change, the signal fluctuates up and down, and data collection is difficult. The omega-type FOLSPR has better detection stability.
4. The detection principle adopted by the invention adopts the specific recognition of the aptamer and the bacteria, so that the aptamer has high affinity and specificity to a sample to be detected.
5. The measuring method adopted by the invention is local surface plasma resonance, the light source is common white light, and the acquired signal is a continuous spectrum, so all reaction processes can be monitored in real time, and any reactant cannot be marked in the reaction processes. Realizes unmarked and real-time monitoring of the reaction.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Fig. 1 is a schematic structural view of an Ω -type optical fiber according to the present invention.
FIG. 2 is a schematic structural diagram of an implementation device of the omega-type FOLSPR sensor in the invention: 5-light source, 6-displacement table, 7-spectrometer, 8-computer, 9-metal bath, and the insertion picture is a schematic diagram of FOLSPR probe for detecting bacteria.
FIG. 3 shows the results of detecting the sensitivity of omega-type FOLSPR in the present invention.
FIG. 4 is a time plot of bacteria in a chicken sample measured using the method of the present invention.
Detailed Description
The following description of the present invention with reference to the drawings is provided to illustrate embodiments of the present invention and to further explain the present invention by way of the embodiments so as to facilitate understanding of the present invention.
Example 1
A preparation method of a novel omega-shaped FOLSPR probe sensor comprises the following steps:
1) preparing an optical fiber; a communication optical fiber having a core diameter of 600 μm and a length of 20cm was used. Polishing the two ends of the optical fiber to a smooth and flat state, burning off the cladding under the condition of flame high temperature, and processing into omega shape.
2) Functionalization of the optical fiber; the optical fiber is subjected to surface modification to achieve the purpose of detection. Optical fiber with removed cladding portion immersed in 30% H2O2And H2SO4And (4) mixing the solution. Ultrasonic washing, oven drying, soaking in 3-APTMS solution at room temperature for 15min, cleaning, and oven drying. Immersing into nanogold (AuNPs) solution, modifying for 15 minutes, drying, and preparing FOLSPR.
30% H used in step 2)2O2With concentrated H2SO4The volume ratio of (A) to (B) is 3: 7, the reaction temperature is 90 ℃, and the soaking time is 30 minutes.
The 1% by volume 3-APTMS solution in step 2) was prepared by dissolving 3-APTMS in a mixture of ethanol and acetic acid in a volume ratio of 5: 2.
After the 3-APTMS solution is soaked in the step 2), the cleaning condition is that 95% ethanol is used for ultrasonic cleaning for 15 minutes, and the water is used for 10 minutes.
3) The synthesis method of AuNPs comprises the following steps: 50ml of HAuCl in the flask4·3H2The aqueous O solution was heated to boiling and then the flask was charged with a solution of trisodium citrate. When the wine red was stable, the mixture was boiled for a further 15 minutes and cooled to room temperature with stirring. HAuCl4·3H2The concentration of O is 0.1mg/mL, and the concentration of trisodium citrate solution is 1% (w/v).
4) And connecting the sulfydryl modified aptamer on the surface of the FOLSPR, and functionalizing the FOLSPR probe. The sequence of the thiol-modified aptamer is:
the oligonucleotide fragment of SH-C6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG is used for detecting salmonella typhimurium, staphylococcus aureus, shigella, escherichia coli and salmonella enteritidis.
Example 2
In this embodiment, the Ω -type FOLSPR probe used for detection is shown in fig. 1, and the sensor detection device is shown in fig. 2. The structure of the detection device mainly comprises a light source 1, a displacement table 2, a spectrometer 3, a computer 4 and a metal bath 5. Light emitted by the light source is transmitted to the FOLSPR probe through the optical fiber, incident photons and free electrons on the surface of the optical fiber generate resonance in the detection part to generate local surface plasma resonance to cause light absorption, the rest light can be transmitted into the spectrometer through the optical fiber, and a generated light absorption signal is transmitted into a computer by the spectrometer to perform real-time monitoring and qualitative and quantitative analysis.
The measurement process is as follows:
detection of FOLSPR refractivity of omega type
1) RI sensitivity analysis of FOLSPR in omega form. And respectively immersing the FOLSPR into 0-20% sucrose solution, measuring the light absorption value, and linearly fitting the change of the light absorption value with the change of the sucrose refractive index to obtain the slope which is the refractive sensitivity. In order to evaluate the RI size of the omega-shaped FOLSPR, the RI of the omega-shaped FOLSPR was compared with that of the U-shaped FOLSPR, and the result was 2.5 times that of the U-shaped FOLSPR, and the result is shown in FIG. 3.
2) The reason why RI sensitivity of omega-shaped FOLSPR in 1) is higher than that of U-shaped FOLSPR is investigated.
3) The specific experimental process in step 2): recording the signal generated by the contact of FOLSPR on the liquid surface as the first point, then recording the next point every 0.5mm of drop, measuring the absorbance value of each point in 0-20% sucrose solution, calculating the RI sensitivity of each point according to the method in 1), and making the change curve of the RI sensitivity along with the change of the immersion depth. Comparing the RI sensitivity of the Ω -shaped FOLSPR with the U-shaped FOLSPR, it is found that the reason why the RI sensitivity of the Ω -shaped FOLSPR is higher is to have more bent regions.
Example 3
Detection of bacteria in chicken samples
40g of chicken was removed, minced, and soaked in 200mL of buffer (10mM Tris-HCl pH 7.4, 5mM KCl, 100mM NaCl, 1mM MgCl2) for 1 h. And adding salmonella typhimurium with different concentrations into the supernatant, and measuring FOLSPR light absorption value signals. And (3) taking the light absorption value at the absorption peak value to make a time curve, and reacting the combination condition of the bacteria and the aptamer in real time. The real-time labeling-free detection of the chicken sample is realized. FIG. 4 is a time plot of bacteria in a chicken sample measured using the method of the present invention.
In conclusion, the invention discloses an omega-shaped optical fiber detection probe, and the optical fiber detection probe in the shape is combined with biosensing to obtain a novel sensor for detecting pathogenic bacteria. The novel omega-shaped optical fiber is processed by adopting flame at high temperature, and the processing process is simple. The FOLSPR probe is assembled by modifying the surface of an optical fiber and connecting nanogold to prepare optical fiber surface plasmon resonance (FOLSPR), connecting aptamer DNA on the surface of the FOLSPR and adopting continuous spectrum, thereby realizing the real-time detection of a biological sample. The performance of the probe is researched, the advantages of the refractive sensitivity of the optical fiber probe are demonstrated, the mechanism causing the sensitivity to be improved is deeply researched, and finally the probe is successfully applied to the detection of pathogenic bacteria. The novel omega-shaped optical fiber sensor has the advantages of simplicity and convenience in operation, high sensitivity, good stability and capability of detecting biological combination in real time.
SEQUENCE LISTING
<110> Sichuan university
<120> preparation method of optical fiber sensor and application of optical fiber sensor in bacterial detection
<130>GYKH1486-2019P018092CCZ
<150>2018111748777
<151>2019-10-09
<160>1
<170>PatentIn version 3.5
<210>1
<211>40
<212>DNA
<213>Artificial Sequence
<220>
<223> thiol-modified aptamer
<400>1
tatggcggcg tcacccgacg gggacttgac attatgacag 40
Claims (10)
1. An optical fiber, characterized by: the structure is as follows: the cladding-containing part 2 is characterized by comprising an exposed part 1 and a cladding-containing part 2, wherein the exposed part 1 comprises an arc section 3 larger than a semicircle and two straight sections 4, one ends of the two straight sections 4 are respectively connected with two ends of the arc section 3, the connecting part of the straight sections 4 and the arc section 3 is rounded, the distance between the two straight sections 4 is gradually increased from one end connected with the arc section 3 to the other end, and the other end of the straight section 4 is connected with the cladding-containing part 2; preferably, the core diameter of the optical fiber is 600 μm, and the length of the optical fiber is 10-50 cm.
2. A method of making the optical fiber of claim 1, wherein: it comprises the following steps: taking an optical fiber, polishing two ends of the optical fiber to a smooth and flat state, burning off a cladding of a part needing to be exposed under the condition of high temperature of flame, and processing and molding.
3. Use of the optical fiber of claim 1 for the preparation of an optical fiber surface plasmon resonance probe.
4. An optical fiber surface plasmon resonance probe, characterized in that: attaching an adapter for inspection to the surface of the bare portion of the optical fiber of claim 1; preferably, the aptamer is a thiol-modified aptamer; more preferably, the thiol-modified aptamer is: SH-C6-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG.
5. A method for preparing the optical fiber surface plasmon resonance probe of claim 4, characterized in that: it comprises the following steps:
1) the bare portion of the optical fiber was immersed in 30% H2O2And concentrated H2SO4Ultrasonic washing and drying are carried out in the mixed solution;
2) then immersing the bare part of the optical fiber into a 3-APTMS solution, cleaning and drying;
3) then immersing the bare part of the optical fiber into the nano gold solution, modifying for 10-40 minutes, preferably 15 minutes, and drying;
4) finally, connecting a mercapto-modified aptamer on the surface of the exposed part of the optical fiber to obtain an optical fiber surface plasma resonance probe;
preferably, the first and second electrodes are formed of a metal,
in step 1), 30% H2O2With concentrated H2SO4The volume ratio of the components is 3: 7, the soaking temperature is 90 ℃, and the soaking time is 30 minutes;
in the step 2), the preparation method of the 3-APTMS solution is to dissolve the 3-APTMS in a mixture of ethanol and acetic acid with the volume ratio of 5: 2, and the concentration of the 3-APTMS solution is 1 percent (v/v);
in the step 2), the mixture is subjected to ultrasonic cleaning for 15 minutes and water cleaning for 10 minutes in 3-APTMS solution at room temperature for 10-20 minutes, preferably 15 minutes;
in the step 3), the synthesis method of the nano gold is to mix 50mL of HAuCl in a flask4·3H2Heating the O aqueous solution to boiling, adding a trisodium citrate solution into the flask, continuing to boil the mixture for 15 minutes when the red color of the wine is stable, and cooling to room temperature under stirring; wherein, HAuCl4·3H2The concentration of O is 0.1mg/mL, and the concentration of trisodium citrate solution is 1% (w/v);
in the step 3), the soaking conditions in the nano gold solution are as follows: at room temperature, 10-40min, preferably 15min, and drying directly without cleaning.
6. Use of the fiber surface plasmon resonance probe of claim 4 for the preparation of a fiber surface plasmon resonance probe sensor.
7. An optical fiber surface plasmon resonance probe sensor, characterized in that: it comprises the optical fiber surface plasma resonance probe, a tungsten lamp light source and an optical fiber spectrometer of claim 4.
8. Use of the fiber surface plasmon resonance probe sensor of claim 7 for pathogen detection.
9. Use according to claim 8, characterized in that: the pathogenic bacteria are salmonella typhimurium, staphylococcus aureus, shigella, escherichia coli and salmonella enteritidis; preferably, the salmonella typhimurium concentration is changed to 5 × 102To 1X 108CFU/mL, Staphylococcus aureus, Shigella, Escherichia coli, Salmonella enteritidis, as control for specific recognition, at a concentration of 106CFU/mL。
10. Use according to claim 8 or 9, characterized in that: the combination time of the pathogenic bacteria and the aptamer is 1h-3h, preferably 1.5 h;
and/or the culture conditions of pathogenic bacteria are as follows: LB medium, 200rpm/min, 37 ℃ overnight, the temperature for pathogen and aptamer binding was 37 ℃.
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